Field of the Invention
This invention generally relates to regenerative cells derived from a wide variety of tissues, and more particularly, to adipose-derived regenerative cells (e.g., stem and/or progenitor cells), methods of using adipose-derived regenerative cells, compositions containing adipose-derived regenerative cells, and systems for preparing and using adipose-derived regenerative cells which are used in the treatment of stroke, including ischemic stroke and hemorrhagic stroke.
Description of the Related Art
The American Heart Association estimates 700,000 people in the United States will have a new or recurrent stroke in 2003, effectively 1 every 45 seconds. The debilitating consequences for those who survive a stroke are substantial; 15-30% of stroke survivors are permanently disabled, 20% require institutional care and in 1999, 1.1 million Americans reported stroke-related functional limitations affecting daily living.
The considerable majority (88%) of stroke cases are the result of ischemia (low tissue oxygen supply) due to blockages to the blood vessels serving the central nervous system (CNS). These blockages arise from narrowing of the vessels due to build up of atherosclerotic plaques usually in combination with occlusion due to entrapment at the narrowed region of small clots of aggregated platelets (thrombus). The remaining 12% of strokes arise from hemorrhagic (bleeding) events (9% intracerebral hemorrhage, 3% subarachnoid hemorrhage) in which a blood vessel within the CNS ruptures leading to mechanical and ischemic damage. The death rate from hemorrhagic stroke is considerably higher than that of ischemic injury; 7.6% of ischemic strokes result in death within 30 days compared to 37.5% of hemorrhagic stroke.
Neuronal damage following damage to the CNS is a routine occurrence and is the result of a number of mechanisms including necrosis due to acute ischemic insult, excitotoxicity, and ongoing inflammatory and apoptotic injury. Specifically, in the center of the infarct region rapid cell death through necrotic processes occurs within minutes of the onset of ischemia. The dependence of the brain on oxidative phosphorylation is such that hypoperfusion rapidly restricts the ability of neurons to maintain ionic gradients and a membrane potential. This inability leads to neuronal depolarization and subsequent activation of voltage-dependent calcium channels and release of excitatory molecules. These events are exacerbated by the failure of energy-dependent reuptake mechanisms leading to further accumulation of excitatory molecules in the extracellular space. This progresses to ion and water efflux and cerebral edema, which can spread through the penumbra due to spreading depolarization.
Numerous therapeutic approaches exist to treat stroke and associated complications. Many of these approaches, however, suffer from a number of limitations. For example, intravenous recombinant tissue plasminogen activator (tPA) initiated within three hours of stroke onset remains the only approved and validated therapeutic option. However, studies have shown that initiation of tPA beyond 90 minutes results in relatively modest benefit. Neuroprotective strategies designed to mitigate this process by modifying ion fluxes and the release, reuptake, and response to excitatory molecules, e.g., agents which scavenge or reduce the action of reactive oxygen species have also been shown to have substantial neuroprotective potential. However, many strategies that appeared promising in animal studies have been disappointing in clinical trials in large part because of the delay in therapy that is necessarily encountered outside of the laboratory setting.
Alternate strategies to reopen the occluded vessel(s) including mechanical clot retrieval and intra-arterial thrombolytic (‘clot-busting’) devices are being developed and tested as are pharmacologic anti-thrombotics such as GPIIb/IIIa inhibitors. However, the ability of these strategies to be effective is limited by the irreversible pathologic processes initiated by the original ischemic event and its ongoing deleterious sequelae. Specifically, reopening of the vessels leading to return of blood flow (reperfusion) is associated with a different form of damage. The newly delivered oxygen is rapidly converted into reactive oxygen species by the environment of the previously ischemic area. Reactive oxygen species result in membrane oxidation, mitochondrial damage and the subsequent initiation of programmed cell death. Apoptosis (programmed cell death) is also associated with ongoing hypoperfusion.
Elucidation of stroke-related progressive injury mechanisms, however, has led to development of anti-apoptotic and pro-angiogenic strategies in which the therapeutic agents are applied no earlier than 3 hours and as much as 24 hours after the initial injury. Growth factors with anti-apoptotic function have also been applied with some success in experimental models. For example, Tsusuki et al have shown that Hepatocyte Growth Factor (HGF) reduces infarct volume. A pro-angiogenic strategy has also shown promise in recent studies.
Another approach has been to harness cells believed to contribute to normal regenerative mechanisms in response to ischemic injury. Thus, mesenchymal stem cells (MSC) and endothelial progenitor cells have been proposed as potential mediators of therapeutic benefit for cerebral ischemia. In these studies bone marrow stromal cells or umbilical cord blood-derived cells were administered in experimental models 24 hours following onset of ischemia. These effects may be mediated by paracrine production of anti-apoptotic and/or angiogenic factors by donor cells or by direct contribution of donor cells to angiogenesis or neurogenesis. Indeed, in the study with cord blood-derived cells donor-derived cells with neuronal markers were detected in the infarct zone.
MSC populations have been shown to be present in one or more of bone marrow, skin, muscle, liver and brain (Jiang et al., 2002b; Alison, 1998; Crosby and Strain, 2001). However, the frequency of MSC in these tissues is low. For example, mesenchymal stem cell frequency in bone marrow is estimated at between 1 in 100,000 and 1 in 1,000,000 nucleated cells (D'Ippolito et al., 1999; Banfi et al., 2001; Falla et al., 1993). Similarly, extraction of stem cells from skin involves a complicated series of cell culture steps over several weeks (Toma et al., 2001) and clinical application of skeletal muscle-derived stem cells requires a two to three week culture phase (Hagege et al., 2003). Thus, any proposed clinical application of stem cells from such tissues requires increasing cell number, purity, and maturity by processes of cell purification and cell culture.
Although cell culture steps may provide increased cell number, purity, and maturity, they do so at a cost. This cost can include one or more of the following technical difficulties: loss of cell function due to cell aging, loss of potentially useful non-stem cell populations, delays in potential application of cells to patients, increased monetary cost, and increased risk of contamination of cells with environmental microorganisms during culture. Recent studies examining the therapeutic effects of bone-marrow derived MSC have used essentially whole marrow to circumvent the problems associated with cell culturing (Horwitz et al., 2001; Orlic et al., 2001; Stamm et al., 2003; Strauer et al., 2002). The clinical benefits, however, have been suboptimal, an outcome almost certainly related to the limited ASC dose and purity inherently available in bone marrow.
Recently, adipose tissue has been shown to be a source of ASCs (Zuk et al., 2001; Zuk et al., 2002). Unlike marrow, skin, muscle, liver and brain, adipose tissue is comparably easy to harvest in relatively large amounts (Commons et al., 2001; Katz et al., 2001b). Furthermore, adipose derived stem cells have been shown to possess the ability to generate multiple tissues in vitro, including bone, fat, cartilage, and muscle (Ashjian et al., 2003; Mizuno et al., 2002; Zuk et al., 2001; Zuk et al., 2002). Thus, adipose tissue presents an optimal source for ASCs for use in regenerative medicine.
Suitable methods for harvesting adipose derived stem cells, however, may be lacking in the art. Existing methods may suffer from a number of shortcomings. For example, the existing methods may lack the ability to optimally accommodate an aspiration device for removal of adipose tissue. The existing methods may also lack partial or full automation from the harvesting of adipose tissue phase through the processing of tissue phases (Katz et al., 2001 a) and/or. The existing methods further may lack volume capacity greater than 100 ml of adipose tissue. The existing methods may yet further lack a partially or completely closed system from the harvesting of adipose tissue phase through the processing of tissue phases. Finally, the existing methods may lack disposability of components to attenuate concomitant risks of cross-contamination of material from one sample to another. In summary, the many prior art methods for harvesting stem cells from adipose tissue do not appear to overcome the technical difficulties associated with harvesting stem cells from skin, muscle, liver and brain described above.
Given the tremendous therapeutic potential of adipose derived stem cells for the treatment of stroke and associated diseases and disorders, there remains a need in the art for systems and methods that are capable of harvesting regenerative cell populations, e.g., stem cells, with increased yield, consistency and/or purity and of doing so rapidly and reliably with a diminished or non-existent need for post-extraction manipulation. Ideally, such a device, system or method would yield regenerative cells in a manner suitable for direct placement into a recipient.